Background: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with
production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal
line produce two populations of cells, characterised as CD11b+CD11c+MHC-II− dendritic-like ‘L-DC’, and CD11b+CD11c+
MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of
these two subsets has not been clearly distinguished.
Results: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T
cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+
T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL10
production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the
proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDClike
cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene
expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype,
while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with
functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell
contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF
production by stroma.
Conclusion: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of
dendritic-like cells. These are here distinguished phenotypically and through gene expression differences.
While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the
cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through
Transwell co-cultures with cell production dependent on M-CSF.
Keywords: Hematopoiesis, Dendritic cell, Regulatory dendritic cells, Regulatory T cellsThis work was supported by project grant #585443 to HO from the National
Health and Medical Research Council of Australia. SP was supported by a
graduate scholarship from the Royal Thai Government. PP was supported by
an Australian National University Graduate Scholarship
